Methods and compositions for the treatment and prevention of cancer

ABSTRACT

The instant invention provides compositions for the treatment of cancer. Specifically, the invention provides polypeptides and nucleic acid molecules comprising tumor-associated embryonic antigens, e.g., OFA-iLRP, and chemoattractant ligands, e.g., a proinflammatory chemokine such as MIP3α/CCL20 or β-defensin mDF2β. The invention further provides cancer vaccines and methods for treating subjects having, or at risk of developing, cancer.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application60/841,927, filed Sep. 1, 2006. The entire contents of theaforementioned application are hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

The basis for the high expectations of cancer immunotherapy is in itsability to eliminate the residual malignant cells and prevent relapse ofthe disease. The simplest method is to induce tumor-specific immunity byimmunizing patients with the antigenic components of their tumors, socalled tumor-associated antigens (TAAs). However, TAAs are often poorlyimmunogenic and their repertoire for immunotherapeutic use is quitelimited. Unlike solid tumors, immunotherapy for B cell malignancies isfurther hampered by lack of well defined TAAs, except for the patient'sunique idiotypic antibody (Id). Although efficacy of the Id vaccinesboth in preclinical studies and phase I-II clinical tests isdemonstrably potent², a broader application of the vaccines may not befeasible due to the unpredictability of their T cell epitopes³, neededfor T cell responses, and the suppressive nature of tumor derived Id inthe absence of continuing T cell help⁴. In addition, Id vaccines have tobe custom tailored and individually produced for each patient. Idiotypicvaccines for some B cell malignancies have been shown to be effectiveboth in animal models^(9;32-34) and in phase I-III clinical trials³⁵.However, a major limitation of this method is not only that the vaccineis individually produced for each patient (see review^(36;37)), but alsothat the T cell epitopes essential for the protection may not always beexpressed on Id.

Recently, the oncofetal Ag-immature laminin receptor 37-kDa protein,OFA-iLRP, was reported to be specifically expressed in different humantumors, such as breast, renal, lung and ovarian cancers, and inhematological malignancies¹. OFA exists in two forms, as the dimerizedhigh-affinity mature 67-kDa mLRP that may act as a cofactor to stabilizethe binding of laminin to cell surface integrins, and the 37-kDaOFA-iLRP, which is not expressed by adult differentiated tissues⁵. Theimmunotherapeutic potential of OFA-iLRP has been recently proposed, asHLA-A2 specific CD8⁺ cells, generated from the peripheral blood ofhealthy donors or cancer patients, lysed OFA-iLRP⁺ acute myeloidleukemia (AML) and chronic lymphocytic leukemia (CLL) cells^(6;7).

Unlike Id, OFA-iLRP is highly evolutionary conserved antigen thatcontains number of CD8⁺ T cell epitopes expressed by human cancercells⁷. Accordingly, a need exists for the development of anti-cancervaccines that are not individually tailored and have broad ability totreat and prevent cancer, and OFA-iLRP may be useful if it can be madeantigenic.

SUMMARY OF THE INVENTION

The inventors of the instant application have developed a novel strategyfor rendering weakly or non-immunogenic self tumor antigens immunogenic.The strategy is based on use of proinflammatory chemokines to deliverantigens to immature DCs through targeting chemokine receptorsdifferentially expressed on APCs^(1;2). Using the technology describedherein, protein or DNA immunizations elicit therapeutic antitumorimmunity against wide variety of tumors, which express non-immunogenicor weakly immunogenic tumor antigens, such as, for example, theembryonic antigen OFA.

Accordingly, the instant invention is based, at least in part, on thediscovery that tumor-associated embryonic antigens, e.g., OFA-iLRP,though non-antigenic alone, are effective for the treatment and/orprevention of cancer when linked to a chemoattractant ligand, e.g., aproinflammatory chemokine such as MIP3α/CCL20 or β-defensin mDF2β.Accordingly, the instant invention provides methods and compositions forthe treatment and prevention of cell proliferative disorders, e.g.,cancer, using the discovered molecules.

In one aspect, the invention provides nucleic acid molecules encoding atumor-associated embryonic antigen and a chemoattractant ligand. In oneembodiment, the tumor-associated embryonic antigen is human or mouseOFA-iLRP. In another embodiment, the chemoattractant ligand is specificfor CCR6, e.g., MIP3α/CCL20 or β-defensin DF2β. In particularembodiments, the chemoattractant ligand is human or murine. In anotherembodiment, the chemoattractant ligand is murine or human EP2C, murineor human β-defensin 1 (MBD1), or a C-terminal fragment of mycobacterialHSP 70.

In a specific embodiment, the invention provides nucleic acid moleculesencoding β-defensin DF2β and OFA-iLRP, or functional fragments thereof.In another specific embodiment, the β-defensin DF2β is human β-defensinDF2β. In yet another specific embodiment, the β-defensin DF2β is murineβ-defensin DF2β. The sequence of one exemplary nucleic acid moleculeencoding β-defensin DF2β and OFA-iLRP is set forth as SEQ ID NO: 1.

In another specific embodiment, the invention provides nucleic acidmolecules encoding MIP3α/CCL20 and OFA-iLRP, or functional fragmentsthereof. In one specific embodiment, the MIP3α/CCL20 is humanMIP3α/CCL20. In yet another specific embodiment, the MIP3α/CCL20 ismurine MIP3α/CCL20. The sequence of one exemplary nucleic acid moleculeencoding MIP3α/CCL20 and OFA-iLRP is set forth as SEQ ID NO:3.

In another specific embodiment, the invention provides nucleic acidmolecules encoding EP2C and OFA-iLRP, or functional fragments thereof.In one specific embodiment, the EP2C is human EP2C. In yet anotherspecific embodiment, the EP2C is murine EP2C. The sequence of oneexemplary nucleic acid molecule encoding EP2C and OFA-iLRP is set forthas SEQ ID NO: 5.

In another specific embodiment, the invention provides nucleic acidmolecules encoding the C-terminal fragment of mycobacterial HSP 70 andOFA-iLRP, or functional fragments thereof. The sequence of one exemplarynucleic acid molecule encoding C-terminal fragment of mycobacterial HSP70 and OFA-iLRP is set forth as SEQ ID NO: 7.

In specific embodiments, the OFA-iLRP is murine OFA-iLRP. In otherspecific embodiments, the OFA-iLRP is human OFA-iLRP.

In another embodiment, the invention provides nucleic acid moleculesencoding a linker polypeptide between the tumor-associated embryonicantigen and the chemoattractant ligand. In another aspect, theembodiment, the invention provides nucleic acid molecules encoding apurification tag, e.g., a myc or his tag. In yet another embodiment, theinvention provides nucleic acid molecules described herein furtherencoding a signal sequence, e.g., the IP 10 signal sequence.

In another aspect, the invention provides vectors comprising the nucleicacid molecules described herein.

In another aspect, the invention provides the nucleic acid moleculesdescribed herein for the treatment or prevention of cancer, e.g.,hematological, breast, renal, lung or. ovarian cancer.

In another aspect, the invention provides polypeptides comprising atumor-associated embryonic antigen and a chemoattractant ligand. In oneembodiment, the tumor-associated embryonic antigen is OFA-iLRP. Inanother embodiment, the chemoattractant ligand is specific for CCR6,e.g., MIP3α/CCL20 or β-defensin mDF2β. In one embodiment, theMIP3α/CCL20 or β-defensin DF2β is human or murine MIP3α/CCL20 orβ-defensin DF2β.

In one embodiment, the chemoattractant ligand is murine or human EP2C,human β-defensin 1 (MBD1), or a C-terminal fragment of mycobacterial HSP70.

In another embodiment, the invention provides polypeptides comprisingβ-defensin DF2β and OFA-iLRP, or functional fragments thereof. In arelated embodiment, the β-defensin DF2β is human β-defensin DF2β. Inanother related embodiment, the β-defensin DF2β is murine β-defensinDF2β. The sequence of one exemplary polypeptide comprising β-defensinDF2β and OFA-iLRP is set forth as SEQ ID NO: 2.

In another embodiment, the invention provides polypeptides comprisingMIP3α/CCL20 and OFA-iLRP, or functional fragments thereof. In a relatedembodiment, the MIP3α/CCL20 is human MIP3α/CCL20. In another relatedembodiment, the MIP3α/CCL20 β is murine MIP3α/CCL20 β. The sequence ofone exemplary polypeptide comprising MIP3α/CCL20 and OFA-iLRP is setforth as SEQ ID NO:4.

In another embodiment, the invention provides polypeptides comprisingEP2C and OFA-iLRP, or functional fragments thereof. In a relatedembodiment, the EP2C is human EP2C. In another related embodiment, theEP2C is murine EP2C. The sequence of one exemplary polypeptidecomprising EP2C and OFA-iLRP is set forth as SEQ ID NO: 6.

In another embodiment, the invention provides polypeptides comprising aC-terminal fragment of mycobacterial HSP 70 and OFA-iLRP, or functionalfragments thereof. The sequence of one exemplary polypeptide comprisinga C-terminal fragment of mycobacterial HSP 70 and OFA-iLRP is set forthas SEQ ID NO: 8.

In certain embodiments, the OFA-iLRP is human OFA-iLRP. In otherembodiments, the OFA-iLRP is murine OFA-iLRP.

In another embodiment, the invention provides polypeptides comprising atumor-associated embryonic antigen and a chemoattractant ligand andfurther comprising a polypeptide linker between the tumor-associatedembryonic antigen and the chemoattractant ligand.

In another embodiment, the invention provides polypeptides comprising atumor-associated embryonic antigen and a chemoattractant ligand andfurther comprising a purification tag, e.g., a myc or his tag.

In another aspect, the instant invention provides a cancer vaccinecomprising the nucleic acid molecules described herein and an adjuvant.In another aspect, the instant invention provides a cancer vaccinecomprising one or more of the polypeptides described herein.

In another aspect, the instant invention also provides methods oftreating a subject having cancer by administering to the subject anucleic acid molecule or polypeptide as described herein, therebytreating the subject. In exemplary embodiments, the cancer is breast,renal, lung, ovarian or a hematological cancer.

In another aspect, the invention provides methods of immunizing asubject against cancer by administering to the subject a nucleic acidmolecule, polypeptide or vaccine as described herein, thereby immunizingthe subject.

The invention also provides a kit comprising a vaccine as describedherein and instructions for use.

The invention also provides a kit comprising a nucleic acid as describedherein and instructions for use.

The invention also provides a kit comprising a polypeptide as describedherein and instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B depict genetic immunizations with constructs expressing mDF2βfusions with non-immunogenic TAAs induce protective anti-lymphoma (A).BALB/c mice (ten per group), immunized with pmDF2β-OFA (closed circle)or pmDF2β-sFv20 (open triangle), were challenged i.p. with 2.5×10⁵ A20lymphoma cells. A separate group of mice were injected with a mixture ofpmDF2β-sFv20 and pMIP3α-OFA (closed square) or mock with PBS (opendiamond). Logrank P-value is for comparison between pmDF2β-OFA orpmDF2β-sFv20 and PBS. A representative experiment of at least threeindependent experiments is shown, all yielding similar results. (B) Miceimmunized with pmDF2β-OFA generate significant OFA-specific IgG1 (opentriangle) and IgG2a (closed triangle). Shown is representative plot ofexperiments of sera mixed five 5 mice per group. No OFA specificantibody was detected in sera of mock immunized mice (open circle, IgG1,and closed circle, IgG2a). Titrated amounts of immune or naïve mousesera were incubated for 1 hour on the same plate coated with 3 μg/ml ofrecombinant TARC-OFA, and the Ig isotypes were determined using goatanti-mouse IgG1- or IgG2a-HRP antibodies (Caltag).

FIGS. 2A-B demonstrate that vaccine induces a T cell response. (A)Splenocytes from mice immunized with pMIP3α-OFA or with the iLR₅₈₋₆₆peptide/IFA specifically lyse A20 lymphoma cells (pMIP3α-OFA/A20 and OFApeptide/A20), but not HLA-matched (H-2K^(d)) but OFA⁻ MOPC315(pMIP3α-OFA/MOPC315 and OFA peptide/MOPC315, or mismatched (H-2^(b))EL-4 (pMIP3α-OFA and OFA peptide/EL4) tumor cells. Control splenocytesfrom mice injected with PBS or immunized with OFA fusions with a mutantMIP3α (pMIP3α-D-OFA), which could not bind CCR6, failed to lyse eitherof cells. Shown here is percentage of cytotoxicity (Y-axis) of tworepresentative and independent experiments with similar results,performed in triplicates. X-axis is effector:target ratio (E:T) of cellsused. (B) Tumor protection requires presence of the OFA-specificeffector CD8⁺ T cells. Mice were immunized with pMIP3α-OFA plasmid asabove and randomly allocated (ten per group) to treatment with anti-CD8mAb GK2.43, anti-CD4 mAb GK1.5, or normal rat IgG. P-values refer tocomparison between anti-CD8 mAb and IgG injected groups. Flow cytometryanalysis of splenocytes from normal mice treated with these mAb inparallel one and two weeks after treatment confirmed a >90% depletion ofthe appropriate subset with normal levels of the other subset (data notshown).

FIGS. 3A-C. (A) Chemoattractants facilitate the CCR6-mediated uptake,processing and presentation of OFA to MHC Class I molecules. NaïveBALB/C mouse iDCs (target cells) were incubated overnight with 100 ng/mlMIP3α-OFA or mDF2β-OFA. Then, after extensive washings and irradiation,they were co-cultured with immune effector splenocytes from BALB/C mice(immunized with the iLR₅₈₋₆₆ peptide/IFA) and IFN-γ release was measuredafter overnight incubation. Effector cell specificity was validatedusing splenocytes pulsed with 1 μg/ml of the iLR₅₈₋₆₆ (OFA peptide) orMOPC315 peptides (irrelevant peptide); or incubating with OFA⁺ A20lymphoma or OFA⁻ MOPC315 tumor cells. Control DCs treated with MIP3αfused with an irrelevant tumor antigen or MC148-D-mOFA (data not shown)or mixture of untreated effector cells with splenocytes (E+T) failed tostimulate T cells. Some iDC were also treated in presence of 0.4 Msucrose, or pertussis toxin (PTX), or chloroquine, or brefeldin A, orlactacystin. P-values refer to comparisons after treatment withchlroquine. (B) Co-localization study. To enable internalization, thepre-chilled on ice cells were placed at 37° C. for the time indicated bythe column headings. Green, MIP3α-fusions stained with anti-myc mAb 1.9μg/mL and goat anti-mouse Alexa 488 2 μg/mL. Red fluorophore, Alexa 568conjugated to goat anti-rabbit IgG, specific for either clathrin (topraw), LAMP (middle row) and proteasomes (bottom row). Merged signal isyellow. Transmission light image is of the 0 min time cell. Scale bar is5 μm (white rectangle). (C) Processed OFA is presented on MHC class Imolecules. iDCs were incubated with mDF2β-OFA or MIP3α-OFA in presenceof neutralizing anti-MHC class I (H-2^(d)) or isotype-matched controlantibodies. Same treatment was performed for control iDCs incubated with1 μg/ml the OFA or MOPC315 peptides. P-values refer to comparisons withcontrol Abs. Shown, representative data of at least two (C) and three (Aand B) independent experiments yielding similar results.

FIG. 4 depicts treatment with pMIP3α-OFA eradicates established A20lymphoma. BALB/c mice (ten mice per group) bearing A20 lymphoma weretreated immunizing with pMIP3α-OFA or pHsp70-OFA. Control mice were mocktreated with PBS or electroporated with pMIP3α-D-OFA. Tumor freesurvival was followed for 100 days post tumor challenge. The data shownis representative of four independent experiments which yielded similarresults. P-value refers to comparison with pMIP3α-D-OFA.

FIG. 5 demonstrates that chemokine or defense fusion proteins are takenup, processed and presented by APCs in vitro via chemokine receptorutilizing MHC class II pathway. Titrated amounts of protein (shown inng/ml), 91-101 peptide or an irrelevant peptide derived from A20lymphoma VL chain were incubated with BALB/c mice immature DCs. APCswere then washed, irradiated and placed in culture with epitope-specific7A10B2 T cell line for 48 hrs, and IFNγ was assayed in culturesupernatants. Control treatment groups were immature DCs or matured byovernight treatment with LPS (10 ng/ml) DCs were pulsed with 0.2 μg/ml91-101 peptide, or with 10 μg/ml irrelevant peptide.

FIGS. 6A-B demonstrate that chemokine fusion enables tumor antigens tobe efficiently cross-presented, i.e. processed and presented to MHCclass I. The intracellular trafficking of Chemokine receptors isdependent on clathrin-associated vesicles (since inhibited with sucrose)and G-protein signaling (inhibited with peruses toxin, PTX) (A).Specificity of effector cells was tested on iDC pulsed with hgp100₂₅₋₃₃peptide, or control A20 peptide, or mixing with cells such as B16melanoma (H-2^(b)), EL4 (H-2^(b)), and A20 (H-2^(d)). iDC were treatedwith 0.1 μg/ml chemokine proteins fused with gp100 in the presence orabsence of various pharmacological inhibitors (μM) of intracellularorganelle trafficking, such as leupeptin and chloroquine (forendosomal-lysosomal), or brefeldin A (vesicle transport between the ERand Golgi). Titrated doses of lactacystin (a specific proteasomalinhibitor, shown in μM), a used to test for cytosolic processing (B).

FIG. 7 demonstrates that cross-presentation of chemokine fusion vaccinesrequires TAP-1 machinery. Immature DC derived from TAP-1 knockout (TAPKO) or wild type C57BL/6 mice were incubated with 0.1 μg/ml eitherMIP3α-gp100 or the gp100 protein alone and tested for their ability tostimulate gp 100-specific T cells derived from pmel-1 mice, asdescribed. Control APC were treated with the active gp 100 peptide,hgp100₂₅₋₃₃, or irrelevant A20 peptides. IFN-γ release was measured inthe supematants of cells cultured for 24 hours by ELISA.

FIG. 8 demonstrates that chemokine fusion vaccination elicits protectiveanti-tumor responses in C57BL/6 mice. Ten mice per group were gene-gunimmunized three times with pMIP3α-gp100, pMIP3α-D-gp100 (a fusion with amutated MIP3α which can not bind to CCR6) or PBS. Two weeks after thelast immunization, mice were challenged s.c. with a lethal dose of B16tumor cells. Tumor growth suppression was subsequently assessed and micewith tumor greater than 400 mm² were euthanized. The data shown isrepresentative of two independent experiments which yielded similarresults. P-value is 0.02.

FIG. 9 demonstrates that tumor protection requires secretion ofchemotactic fusion protein

FIG. 10 demonstrates that antibody responses to the same antigen dependon a type of chemokine used. Mice were gene gun immunized with DNAconstructs expressing non-immunogenic tumor antigen (sFv38) fused withvarious chemokines.

FIGS. 11A-B demonstrate that immunizations with viral chemokine carriersinduce antitumor protection. Ten per group C3H mice were gene gun withplasmids indicated immunized three times in every two weeks, and twoafter, mice were i.p. challenged with 10× lethal dose (3000 cells) of38C13.

FIGS. 12A-B depict the results of experiments demonstrating CCR6 vs.CCR7: MIP3α fusion constructs elicit antitumor protection, although bothSLC and MIP3α fusions generate anti-Id Abs.

FIG. 13 demonstrates that injection of plasmid DNA encoding iDCchemo-attractant fusions elicit therapeutic antitumor immunity.

FIGS. 14.1-14.14 depict SEQ ID NOs:1-32.

FIGS. 15A-D depict eradication of A20 lymphoma promote long-term Tcell-mediated memory that protects mice from re-challenge with A20lymphoma. (A) sixteen mice that were free of tumors for about 9 months(open circles) and control ten age-matched naïve BALB/C mice (closedcircles) were challenged with A20 lymphoma cells. P-value refers tocomparison with control mice. (B) In parallel, splenocytes of long-termsurvivor mice (E, effector cells) were in vitro stimulated for one weekon DCs pused with OFA-peptide and tested against target cells (T), suchas A20, 4T 1, and B 16 tumors, at indicated ration (T:E). Shown,percentage of cytotoxicity (Y-axis) of a representative experimentperformed in triplicate. (C) OFA-iLRP is expressed on the surface of A20lymphoma and B16 melanoma cells, but not 4T1 tumor cells. OFA expressionwas determined with Alexa-488-conjugated anti-OFA mAB (bold lines) vs.control Alexa-488-conjugated isotype-matched AB. (D) Mice that survivedA20 tumor challenge (A20-survivor+4T1, see also A) or control BALB/cmice (HBs+4T1) immunized with control constructs expressing HbsAg werere-challenged with 4T1 tumor cells.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention is based, at least in part, on the discovery thatnon-immunogenic tumor antigens, e.g., OFA-iLRP, can be renderedimmunogenic by using a chemoattractant ligand, e.g., a proinflammatorychemokine. In a preferred embodiment, the tumor antigen andchemoattractant ligand are expressed as a fusion polypeptide or areencoded by a single nucleic acid molecule. These molecules are useful inthe prevention and treatment of cell proliferative disorders, e.g.,cancer. Accordingly, the instant invention provides polypeptides,nucleic acid molecules, vectors, host cells, vaccines, kits and methodsof treating or preventing cancer.

Molecules of the Invention

The present invention provides fusion molecules, e.g., moleculescomprising a tumor antigen and chemoattractant ligand. The tumor antigenand chemoattractant ligand are optionally attached by a linker, e.g., apeptide or non-peptide linker. The invention provides polypeptidescomprising a tumor antigen and chemoattractant ligand and nucleic acidmolecules encoding a tumor antigen and chemoattractant ligand. Incertain embodiments, the molecules comprise fragments of the tumorantigen and/or the chemoattractant ligand, wherein the fragments areeffective to achieve the desired biological effect.

Exemplary tumor antigen are those that are expressed in embryonic tissuebut not in mature tissue. An exemplary tumor antigen useful in themethods and compositions of the invention is the 37 kD oncofetalAg-immature laminin receptor (OFA-iLRP) (SEQ ID NO:31).

Exemplary chemoattractant ligands include proinflammitory chemokines.Specific exemplary chemoattractant ligands include chemoattractantligands specific for CCR6, e.g., MIP3α/CCL20 or β-defensin DF2β. Furtherchemoattractant ligands include EP2C, β-defensin 1 (MBD1), or aC-terminal fragment of mycobacterial HSP 70. For all chemoattractantligands other than mycobacterial HSP 70, the chemoattractant can behuman or murine. The sequence of all the exemplary chemoattractantligands set forth herein are set forth in the sequence of the exemplarypolypeptides and nucleic acid molecules set forth herein.

One of skill in the art can identify chemoattractant ligands andunderstands that homologues and orthologues of these molecules will beuseful in the methods and compositions of the instant invention.Moreover, variants and biologically active fragments of these ligandsare useful in the methods of the invention.

The polypeptides of the invention may be assembled post-translationally,i.e., the tumor antigen and chemoattractant ligand can be covalentlylinked after being synthesized, or expressed, separately. Alternatively,the tumor antigen and chemoattractant ligand can be expressedrecombinantly as one polypeptide.

The polypeptides of the invention may further comprise a polypeptidelinker located between the tumor antigen and chemoattractant ligand. Thepolypeptides of the invention may further comprise one or morepurification tags, e.g., a myc or histidine tag. Finally, thepolypeptides of the invention may comprise a signal sequence to directthe location of the polypeptide.

The invention also provides nucleic acid molecules encoding a tumorantigen and chemoattractant ligand such as those described herein.Moreover, the nucleic acid molecules may further encode a polypeptidelinker located between the tumor antigen and chemoattractant ligand. Thenucleic acid molecules of the invention may further encode a signalsequence to direct the location of the polypeptide. The nucleic acidmolecules of the invention may further encode a purification tag, e.g.,a myc or histidine tag.

The invention also provides vectors, e.g., expression vectors,containing a nucleic acid molecule of the invention. As used herein, theterm “vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid molecule to which it has been linked. One type ofvector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments can be ligated. Another type ofvector is a viral vector, wherein additional DNA segments can be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “expression vectors”. In general, expression vectors are oftenin the form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid molecule of the invention in a form suitable for expression of thenucleic acid molecule in a host cell, which means that the recombinantexpression vectors include one or more regulatory sequences, selected onthe basis of the host cells to be used for expression, which isoperatively linked to the nucleic acid sequence to be expressed. Withina recombinant expression vector, “operably linked” is intended to meanthat the nucleotide sequence of interest is linked to the regulatorysequence(s) in a manner which allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in ahost cell when the vector is introduced into the host cell). The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcells and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences). Itwill be appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, andthe like. The expression vectors of the invention can be introduced intohost cells to thereby produce proteins or peptides, including fusionproteins or peptides, encoded by nucleic acids as described herein(e.g., fusion molecules comprising a chemokine receptor ligand and atoxin moiety).

The recombinant expression vectors of the invention can be designed forexpression of the polypeptides of the invention in prokaryotic oreukaryotic cells. For example, the polypeptides can be expressed inbacterial cells such as E. coli, insect cells (using baculovirusexpression vectors) yeast cells or mammalian cells. Suitable host cellsare discussed further in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret a, Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a residentprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerivisae includepYepSec1 (Baldari, et al., (1987) EMBO J 6:229-234), pMFa (Kudjan andHerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ(InVitrogen Corp, San Diego, Calif.).

Alternatively, the nucleic acid molecules of the invention may be usedto express polypeptides in insect cells using baculovirus expressionvectors. Baculovirus vectors available for expression of proteins incultured insect cells (e.g., Sf9 cells) include the pAc series (Smith etal. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J 8:729-733) andimmunoglobulins (Banedji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

Another aspect of the invention pertains to host cells into which anucleic acid molecule encoding a polypeptide of the invention isintroduced within a recombinant expression vector or a nucleic acidmolecule containing sequences which allow it to homologously recombineinto a specific site of the host cell's genome. The terms “host cell”and “recombinant host cell” are used interchangeably herein. It isunderstood that such terms refer not only to the particular subject cellbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, apolypeptide of the invention can be expressed in bacterial cells such asE. coli, insect cells, yeast or mammalian cells (such as Chinese hamsterovary cells (CHO) or COS cells). Other suitable host cells are known tothose skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAF-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratorymanuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding the polypeptide of the invention or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) the polypeptidesof the invention. Accordingly, the invention further provides methodsfor producing polypeptides using the host cells of the invention. In oneembodiment, the method comprises culturing the host cell of theinvention (into which a recombinant expression vector encoding apolypeptide of the invention has been introduced) in a suitable mediumsuch that a polypeptides of the invention is produced. In anotherembodiment, the method further comprises isolating the polypeptide fromthe medium or the host cell.

The host cells of the invention can also be used to produce non-humantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichcoding sequences have been introduced. Such host cells can then be usedto create non-human transgenic animals in which exogenous sequences havebeen introduced into their genome or homologous recombinant animals inwhich endogenous sequences have been altered. As used herein, a“transgenic animal” is a non-human animal, preferably a mammal, morepreferably a rodent such as a rat or mouse, in which one or more of thecells of the animal includes a transgene. Other examples of transgenicanimals include non-human primates, sheep, dogs, cows, goats, chickens,amphibians, and the like.

Methods of Making the Molecules of the Invention

As described above, molecules of the invention may be made recombinantlyusing the nucleic acid molecules, vectors, and host cells describedabove.

Alternatively, the tumor antigen and chemoattractant ligand can be madesynthetically, or isolated from a natural source and linked togetherusing methods and techniques well known to one of skill in the art.

Further, to increase the stability or half life of the fusion moleculesof the invention, the peptides may be made, e.g., synthetically orrecombinantly, to include one or more peptide analogs or mimmetics.Exemplary peptides can be synthesized to include D-isomers of thenaturally occurring amino acid residues to increase the half life of themolecule when administered to a subject.

Pharmaceutical Compositions

The nucleic acid and polypeptide fusion molecules (also referred toherein as “active compounds”) of the invention can be incorporated intopharmaceutical compositions. Such compositions typically include thenucleic acid molecule or protein, and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” includes solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Supplementaryactive compounds can also be incorporated into the compositions.

Pharmaceutical compositions of the instant invention may also includeone or more other active compounds. Alternatively, the pharmaceuticalcompositions of the invention may be administered with one or more otheractive compounds. Other active compounds that can be administered withthe pharmaceutical compounds of the invention, or formulated into thepharmaceutical compositions of the invention, include, for example,anticancer compounds.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Preferred pharmaceutical compositions of the invention are those thatallow for local delivery of the active ingredient, e.g., deliverydirectly to the location of a tumor.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The protein or polypeptide can be administered onetime per week for between about 1 to 10 weeks, preferably between 2 to 8weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. The skilled artisan willappreciate that certain factors can influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and other diseases present.Moreover, treatment of a subject with a therapeutically effective amountof a polypeptide or nucleic acid molecule can include a single treatmentor, preferably, can include a series of treatments.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack,kit or dispenser together with instructions, e.g., written instructions,for administration, particularly such instructions for use of the activeagent to treat against a disorder or disease as disclosed herein,including an autoimmune disease or disorder, treatment in connectionwith an organ or tissue transplant, as well as other diseases ordisorders with an autoimmune component such as AIDS. The container,pack, kit or dispenser may also contain, for example, a fusion molecule,a nucleic acid sequence encoding a fusion molecule, or a fusion moleculeexpressing cell.

Methods of Treatment

The compositions disclosed herein may be useful in the treatment orprevention of cancer.

The term “cancer” includes malignancies characterized by deregulated oruncontrolled cell growth, for instance carcinomas, sarcomas, leukemias,and lymphomas. The term “cancer” includes primary malignant tumors,e.g., those whose cells have not migrated to sites in the subject's bodyother than the site of the original tumor, and secondary malignanttumors, e.g., those arising from metastasis, the migration of tumorcells to secondary sites that are different from the site of theoriginal tumor.

The term “leukemia” includes malignancies of the hematopoietic cells ofthe bone marrow. Leukemias tend to proliferate as single cells. Examplesof leukemias include acute myeloid leukemia (AML), acute promyelocyticleukemia, chronic myelogenous leukemia, mixed-lineage leukemia, acutemonoblastic leukemia, acute lymphoblastic leukemia, acutenon-lymphoblastic leukemia, blastic mantle cell leukemia, myelodyplasticsyndrome, T cell leukemia, B cell leukemia, and chronic lymphocyticleukemia. Preferred leukemias include T cell malignancies, e.g., T cellleukemia and myeloma.

The invention provides therapeutic methods and compositions for theprevention and treatment of cancer and for the administration of avaccine to a subject.

In one embodiment, the present invention contemplates a method oftreatment, comprising: a) providing, i.e., administering: i) a mammalianpatient particularly human who has, or is at risk of developing, cancer,ii) one or more molecules of the invention.

The term “at risk for developing” is herein defined as individuals withfamilial incidence of, for example, cancer.

The present invention is also not limited by the degree of benefitachieved by the administration of the fusion molecule. For example, thepresent invention is not limited to circumstances where all symptoms areeliminated. In one embodiment, administering a fusion molecule reducesthe number or severity of symptoms of cancer. In another embodiment,administering of a fusion molecule may delay the onset of symptoms.

Typical subjects for treatment in accordance with the individualsinclude mammals, such as primates, preferably humans. Cells treated inaccordance with the invention also preferably are mammalian,particularly primate, especially human. As discussed above, a subject orcells are suitably identified as in needed of treatment, and theidentified cells or subject are then selected for treatment andadministered one or more of fusion molecules of the invention.

The treatment methods and compositions of the invention also will beuseful for treatment of mammals other than humans, including forveterinary applications such as to treat horses and livestock e.g.cattle, sheep, cows, goats, swine and the like, and pets such as dogsand cats.

For diagnostic or research applications, a wide variety of mammals willbe suitable subjects including rodents (e.g. mice, rats, hamsters),rabbits, primates and swine such as inbred pigs and the like.Additionally, for in vitro applications, such as in vitro diagnostic andresearch applications, body fluids (e.g., blood, plasma, serum, cellularinterstitial fluid, saliva, feces and urine) and cell and tissue samplesof the above subjects will be suitable for use.

Vaccines

The preparation of vaccine compositions that contain the nucleic acidmolecules or polypeptides of the invention as an effective ingredient isknown to one skilled in the art. Typically, such vaccines are preparedas injectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to infectioncan also be prepared. The preparation can also be emulsified, or theprotein encapsulated in liposomes. The active immunogenic ingredientsare often mixed with carriers which are pharmaceutically acceptable andcompatible with the active ingredient. The term “pharmaceuticallyacceptable carrier” refers to a carrier that does not cause an allergicreaction or other untoward effect in subjects to whom it isadministered. Suitable pharmaceutically acceptable carriers include, forexample, one or more of water, saline, phosphate buffered saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,and/or adjuvants which enhance the effectiveness of the vaccine.Examples of adjuvants which may be effective include but are not limitedto: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,referred to as nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dip-almitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosporyl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion. Other examples of adjuvants include DDA(dimethyldioctadecylammonium bromide), Freund's complete and incompleteadjuvants and QuilA. In addition, immune modulating substances such aslymphokines (e.g., IFN-g, IL-2 and IL-12) or synthetic IFN-g inducerssuch as poly I:C can be used in combination with adjuvants describedherein.

Vaccine compositions of the present invention may be administeredparenterally, by injection, for example, either subcutaneously orintramuscularly. The vaccine compositions can further be delivered by agene gun. Additional formulations which are suitable for other modes ofadministration include suppositories, and in some cases, oralformulations or formulations suitable for distribution as aerosols. Forsuppositories, traditional binders and carriers may include, forexample, polyalkylene glycols or triglycerides; such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5 to 10%, preferably I to 2%. Oral formulations include such normallyemployed excipients as, for example, pharmaceutical grades of mannitol,lactose, starch magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, and the like. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10% to 95% of effective ingredient,preferably 25 to 70%.

The nucleic acid molecules and proteins of the present invention can beformulated into the vaccine compositions as neutral or salt forms.Pharmaceutically acceptable salts include the acid addition salts(formed with free amino groups of the peptide) and which are formed withinorganic acids such as, for example, hydrochloric or phosphoric acids,or with organic acids such as acetic, oxalic, tartaric, maleic, and thelike. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

Vaccine compositions are administered in a manner compatible with thedosage formulation, and in such amount as will be prophylacticallyand/or therapeutically effective. The quantity to be administereddepends on the subject to be treated, including, e.g., capacity of thesubject's immune system to synthesize antibodies, and the degree ofprotection or treatment desired. Suitable dosage ranges are of the orderof several hundred micrograms effective ingredient per vaccination witha range from about 0.01 to 10 mg/kg/day, preferably in the range fromabout 0.1 to 1 mg/kg/day. Suitable regiments for initial administrationand booster shots are also variable but are typified by an initialadministration followed by subsequent inoculations or otheradministrations. Precise amounts of effective ingredient required to beadministered depend on the judgment of the practitioner and may bepeculiar to each subject. It will be apparent to those of skill in theart that the therapeutically effective amount the vaccine of thisinvention will depend, inter alia, upon the administration schedule, theunit dose of antigen administered, whether the vaccine is administeredin combination with other therapeutic agents, the immune status andhealth of the recipient, and the therapeutic activity of the particularvaccine.

The vaccine can be given in a single dose schedule, or preferably in amultiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination can include 1 to 10 separate doses,followed by other doses given at subsequent time intervals required tomaintain and or reinforce the immune response, for example, at 1 to 4months for a second dose, and if needed, a subsequent dose(s) afterseveral months. Periodic boosters at intervals of 1 to 5 years, usually3 years, are desirable to maintain the desired levels of protectiveimmunity.

Immunization protocols have used adjuvants to stimulate responses formany years, and as such adjuvants are well known to one of ordinaryskill in the art. Some adjuvants affect the way in which antigens arepresented. For example, the immune response is increased when proteinantigens are precipitated by alum. Emulsification of antigens alsoprolongs the duration of antigen presentation.

In one aspect, an adjuvant effect is achieved by use of an agent such asalum used in about 0.05 to about 0.1% solution in phosphate bufferedsaline. Alternatively, the antigen is made as an admixture withsynthetic polymers of sugars used as an about 0.25% solution. Adjuvanteffect may also be made by aggregation of the antigen in the vaccine byheat treatment. Aggregation by reactivating with pepsin treated (Fab)antibodies to albumin, mixture with bacterial cell(s) such as C. parvumor an endotoxin or a lipopolysaccharide components of Gram-negativebacteria, emulsion in physiologically acceptable oil vehicles such asmannide mono-oleate (Aracel A) or emulsion with a 20% solution of aperfluorocarbon used as a block substitute also may be employed.

Various polysaccharide adjuvants may also be used. For example, the useof various pneumococcal polysaccharide adjuvants on the antibodyresponses of mice has been described. The doses that produce optimalresponses, or that otherwise do not produce suppression, should beemployed as indicated. Polyamine varieties of polysaccharides areparticularly preferred, such as chitin and chitosan, includingdeacetylated chitin.

EXAMPLES

It should be appreciated that the invention should not be construed tobe limited to the examples that are now described; rather, the inventionshould be construed to include any and all applications provided hereinand all equivalent variations within the skill of the ordinary artisan.

Materials and Methods Fusion Gene Cloning and Protein Production

Generation of DNA vaccine constructs expressing murine MIP3α/CCL20,murine β-defensin 2 (mDF2β) and Hsp70 fused with tumor antigens(OFA-iLRP or sFv20) was previously described^(8, 9). Hsp70 cDNA was agenerous gift from Dr. Thomas Lehner (Guy's Hospital, London, UK).Murine OFA-iLRP, (OFA, GeneBank # AF140348) was cloned from murine Bcell A20 lymphoma (American Type Culture Collection, (ATCC) Manassas,Va.). All constructs were verified by the DNA sequencing (FidelitySystems, Inc., Gaithersburg, Md.). To generate the DNA vaccine, thechemokine-OFA was cloned in pVAX1 plasmid (Invitrogen).Chemoattractant-OFA proteins were produced from IPTG-induced BL21(DE3)cells (Stratagene) using bacterial expression vector pET11d (Stratagene)and purified (>90% purity) from inclusion bodies as describedpreviously^(8,15). The peptides iLR₅₈₋₆₆ (LLLAARAIV)⁶, MOPC-315 Ig₉₁₋₁₀₁(ALWFRNHFVFGGGTK)¹⁶ were all synthesized by Peptide Technologies(Washington, D.C.) to a purity >99% by HPLC and amino acid analysis.

Cell Lines

The A20 B cell lymphoma (H-2^(d), OFA-iLRP positive), MOPC315plasmacytoma (H-2^(d), OFA-iLRP negative) and EL-4 thymoma (H-2^(b),OFA-iLRP positive) cell lines were purchased from ATCC. The B6/129macrophage cell line (H-2^(d), CCR6 positive by FACS analysis) was agenerous gift from Dr. Howard Young (NCI, MD). Murine bone marrow(BM)-derived DC preparation was previously described¹⁷. Cells used onday 4-5 of cultivation, that usually yields iDCs¹¹.

Immunizations of Mice

All animals were bred or housed at the National Institute of Aginganimal facility, Baltimore, Md. Animal care was provided in accordancewith the procedures outlined in a Guide for the Care and Use ofLaboratory Animals (NIH Publication No. 86-23, 1985). For tumorprotection study, six- to eight-week old female BALB/C mice (ten pergroup) were immunized three times every two weeks by electroporating 25μg DNA in 50 μl endo-free water intradermally (i.d.) into the base oftail using 4 mm-gapped electrodes and PA4000 electric pulse generator(Cyto Pulse Sciences, Inc., Linthicum, Md.) at the following settings: 2pulses at 450V, 0.125 S and 0.05 mS. Two weeks after the lastimmunization, mice were challenged i.p. with 2×10⁵ A20 lymphoma cellsand mice were followed for tumor survival. For therapy studies, six- toeight-week old female BALB/C mice (ten per group) were challenged i.p.with 2×10⁵ A20 lymphoma cells at day 0, and then immunized with DNAconstructs at days 3, 8 and 18. Differences in survival between groupswere determined by non-parametric logrank test (BMDP statisticalsoftware, Los Angeles).

Preparation of Immune Effector Cells, In Vitro Activation of T Cells

Mice were vaccinated s.c. twice at 3-wk intervals with 10 μg humaniLR₅₈₋₆₆ peptide emulsified in 100 μl incomplete Freund's adjuvant(IFA). Three weeks after the second vaccination, splenocytes werecultured with 20 IU/ml rhIL-2 and 1 μg/ml corresponding peptide(irrelevant MOPC-315 Ig₉₁₋₁₀₁ or iLR₅₈₋₆₆, respectively) and used ondays 5-7 after the initiation of the culture.

In Vivo T Cell Subset Depletions.

In vivo antibody depletions started 2 weeks after vaccination bytreatment with three i.p. doses of 400 μg anti-CD8 mAb GK 2.43 oranti-CD4 mAb GK1.5 (NCI-FCRDC, Frederick, Md.), or normal rat IgG(Sigma) every other day two weeks after the last immunization, prior totumor challenge. Depletion of lymphocyte subsets was assessed 1 weekafter final treatment by flow cytometry analysis of splenocytes fromnormal mice treated with these mAb in parallel⁸.

Chemokine Receptor Binding

The ligand binding-internalization assays were performed with iDC orsplenocytes (1×10⁵) blocked with mouse serum in PBS containing 2% BSA.Fusion proteins (10-50 μg/ml) were incubated in complete medium for 1 hat 37° C. or at 4° C. To detect bound proteins, the cells were incubatedwith anti-c-myc mAb or isotype-matched, purified mouse IgG1, followedwith a-mouse Ig-FITC mAb incubation (Jackson ImmunoResearch Laboratory,Bar Harbor, Me.) for 20 min each, and then fixed with 1%paraformaldehyde. The binding-internalization was assessed via flowcytometry on a FACScan (Becton Dickinson, Franklin Lakes, N.J.) usingCellQuest software.

Intracellular Antigen Processing

Antigen presenting cells, splenocytes or iDC, from naïve BALB/c micewere incubated overnight with various concentrations of fusion protein(0.01 -1 μg/ml). The treated APCs were subsequently irradiated (2000Rad), washed twice with PBS to remove unbound proteins, and thencocultured for 24-48 h with specific effector cells from the iLR₅₈₋₆₆(or irrelevant MOPC-315 _(Ig) ₉₁₋₁₀₁) peptide immunized mice. Some APCswere treated overnight with chemokine fused with various inhibitors:pertussis toxin (PTX, 2.5 ng/ml), sucrose (0.4M), brefeldin A (500 μM),chloroquine (50, 10 and 1 μM) and lactacystin (50, 10 and 1 μM). Allreagents were purchased from Sigma.

Cytolytic Assay for Immune Splenocytes

Three per group female BALB/C mice were electroporated with plasmidconstructs as described above or s.c. immunized with 10 μg iLR₅₈₋₆₆peptide/IFA twice with two weeks intervals. Splenocytes were in vitrostimulated with 1 μg iLR₅₈₋₆₆ peptide or irrelevant MOPC315 peptide incomplete RPMI 1640 with IL-2 for one week, then were mixed with targetcells (1×10⁴), A20 lymphoma, MOPC315 and EL4. The cytotoxicity aslactate dehydrogenase release (LDH) in the cell supernatants wasmeasured using the Cytotoxicity Detection Kit (Roche) followingmanufacturer's instructions at the sorbance measured at 570 nm with a630 nm reference filter on a plate reader 680XR (Bio-Rad). The averagevalues for wells performed in triplicate were used for calculationsafter the medium controls were subtracted. The percent-specificcytotoxicity was calculated as: percentcytotoxicity=(experimental−effector alone)−target spontaneous/targetmaximum−target spontaneous.

Confocal Microscopy

B6/129 cells (10⁵) were cultured overnight in covered glass bottomdishes (MatTek Corporation, Ashland, Mass., USA) as describedelsewhere¹⁸. The slides were incubated on ice with 25 μg/ml MIP3α-fusionproteins in 10% FBS/RPMI. After two washes in ice-cold PBS, 10% FBS/RPMIwarmed at 37° C. was added and slides were incubated at 37° C. for 0,10, 30, and 60 minutes before fixation with 3.7% formaldehyde for 10 minand permeabilization with 0.2% Triton X-100 for 5 min at RT. Followingprimary Abs were used: anti myc mAb (clone 9E10, Sigma), and rabbitanti-LAMP-1 antibody (H-228) or rabbit anti-Clathrin HC (H-300, bothfrom Santa Cruz Inc., Calif., USA), or rabbit anti-proteasome 20Ssubunit alpha-5 (Affinity BioReagents, Golden, Colo.). The secondaryAbs, goat anti-mouse or goat anti-rabbit IgG, were conjugated to AlexaFluor 488 or Alexa Fluor 568 (Molecular Probes Inc, Oreg., USA). Afterwashing, a drop of Prolong anti-fade reagent (Molecular Probes) wasadded to each slide well, and images were acquired with a 63× objectiveon a Zeiss LSM 410 confocal system and processed using Adobe Photoshop.

Mice vaccinated with MIP3α/CCL20 fused with OFA-iLRP display longlasting CD8 T cell-dependent protective responses. Specifically, immunemice rejected challenge with synergetic tumor cells even after 9 months(see FIG. 15A-D).

Results and Discussion

DNA vaccines expressing OFA fused to chemo-attractants elicit potentanti-A20 lymphoma protection. Embryonic antigen OFA-iLRP (OFA) is anattractive target for cancer immunotherapy, as it is abundantlyexpressed in various malignancies, including murine A20 lymphoma, andnot found in normal adult tissues¹. Initial attempts to induce anti-A20lymphoma responses in nave BALB/C mice immunized with plasmid DNAexpressing OFA failed, due to poor immunogenicity of the antigen.Therefore, to render OFA immunogenic through the CCR6-mediated targetingof iDCs, constructs which expressed OFA fusions with mDF2β (pmDF2β-OFA)or MIP3α/CCL20 (pMIP3α-OFA) were generated. Ten per group naïve BALB/Cmice were immunized with either pmDF2β-OFA or with pmDF2β-sFv20, apositive control construct that encoded mDF2β fusion to A20-specific Igfragment (single chain Fv) shown to be immunogenic⁹. Then, two weeksafter the last immunization, mice were challenged with a lethal dose ofA20 lymphoma cells. Almost all mice mock immunized with PBS succumbed tocancer (PBS, FIG. 1 a). In contrast, mice immunized with pmDF2β-OFA orpmDF2β-sFv20 acquired significant protection against A20 lymphoma(p<0.05, as compared with PBS treated mice, FIG. 1 a). The responserequired targeting of CCR6, as control vaccines that expressed OFA fusedto mutant MIP3α, which did not bind CCR6 due to a single pointmutation¹¹, failed to protect (pMIP3α-D-OFA, see FIG. 4). Thus,pmDF2β-OFA is as potent as the Id vaccine (pmDF2β-sFv20) and inducescomparable protective anti-B cell lymphoma responses. However, unlikeId, OFA-based vaccines would not require individual formulations foreach patient; instead, they might be used for the treatment of anyOFA-expressing cancers.

Tumor protection is not improved by use of multiple TAA-encodingvaccines. Since either of the vaccines that expressed different tumorantigens, pmDF2β-sFv20 or pMIP3α-OFA, elicited comparable responses, wetested whether they would also act additively when used together(pmDF2P-sFv20+pMIP3α-OFA). As shown in FIG. 1 a, mice were protectedagainst A20 lymphoma at almost the same level regardless of whether theywere immunized with the vaccine mixture or with a singleantigen-expressing vaccine (see pmDF2β-sFv20+pMIP3α-OFA vs. pmDF2β-OFAor pmDF2β-sFv20, FIG. 1 a). Thus, immune responses elicited against asingle TAA can be sufficiently high to protect against tumors, and useof additional antigens may not be necessary or beneficial.

Tumor protection depends on induction of effector CD8⁺ T cells. Miceimmunized with pmDF2β-OFA or pMIP3α-OFA generated not only OFA-specificIgG1 antibodies (open triangle, FIG. 1 b), but also significant levelsof IgG2a antibody (closed triangle, FIG. 1 b), indicating that theymight produce Th1 responses¹⁹. Moreover, mice immunized with thevaccines generated cytolytic T cells (CTLs) capable of specific killingof A20 tumor cells in vitro (FIG. 2 a). The CTLs were specific to OFA,as they did not lyse irrelevant HLA-matched MOPC315 cells, which did notexpress OFA (FIG. 2 a). The response was dependent on the ability of thevaccine to target CCR6, since splenocytes from mice immunized with theconstruct expressing OFA fused to a mutant MIP3α (pMIP3α-D-OFA, FIG. 2a) did not kill A20 lymphoma cells. Since mice immunized withpMIP3α-D-OFA were also not protected (FIG. 4), it is tempting tospeculate that the protection was mediated by these CTLs. To study this,CD8⁺ or CD4⁺ effector cells were depleted in mice immunized withpMIP3α-OFA by injecting specific antibodies prior to the challenge withA20 lymphoma cells. Injections of isotype-matched irrelevant IgG(pMIP3α-OFA+IgG, FIG. 2 b), or the depletion of effector CD4⁺ T cells(pMIP3α-OFA+αCD4 Ab, FIG. 2 b) did not have any effects and miceimmunized with pMIP3α-OFA remained protected. In contrast, theprotection was completely abolished in mice that were depleted ofeffector CD8⁺ T cells (pMIP3α-OFA+αCD8 Ab, FIG. 2 b). Taken together,these data clearly indicate that, as we also reported for Id-mediatedanticancer protection⁹, the protection was primarily dependent on theactivation of cellular immunity, particularly effector CD8⁺ T cells, butnot humoral responses despite the fact that both Id and OFA-iLRP areexpressed on the cell surface. Thus, the breadth of the CCR6-targetingchemoattractant-based OFA vaccines is in their ability to elicittumor-specific CD8⁺ cytolytic T cell responses.

The CCR6-targeted OFA is efficiently taken up and cross-presented to MHCclass I molecules. CCR6 would efficiently internalize upon binding withits ligands MIP3α or mDF2β⁹. Similarly, unlike control OFA constructs(OFA alone or fused with mutant chemokines), MIP3α-OFA or mDF2β-OFA weretaken up through CCR6 expressed on murine BM iDC (data not shown),suggesting that the CTL responses observed might be duecross-presentation of the internalized OFA. To test this, naïve BM iDCsfrom BALB/C mice were incubated overnight with nM concentrations ofpurified recombinant MIP3α-OFA or mDF2β-OFA proteins. Then, afterextensive washing and irradiation steps, the cells were mixed withimmune splenocytes from syngeneic mice immunized with the peptideOFA-iLRP₅₈₋₆₆ in IFA, which elicited CTLs capable of specific killing ofA20 lymphoma cells in vitro, but not control HLA-matched MOPC315 cellsthat did not express OFA (FIG. 2 a). The assumption was that, if CCR6mediated cross-presentation, APCs incubated with MIP3α-OFA or mDF2β-OFA,but not free OFA, would stimulate the OFA-iLRP₅₈₋₆₆ peptide-specific Tcells. As shown in FIG. 3 a, only iDCs incubated with as little as 100ng/ml MIP3α- or mDF2β-OFA fusion proteins induced significant IFNγsecretion from the OFA peptide-specific T cells, suggesting thatchemoattractant fused OFA was processed and presented to MHC class Imolecules. Control DCs incubated with MIP3α-OFA or mDF2βsFv20(irrelevant tumor antigen fusions, data not shown) did not stimulate thesplenocytes, ruling out non-specific effects from the chemoattractantsused. Thus, these data indicate that MIP3α-OFA was efficientlycross-presented, which involved an active receptor-mediated process, aspertussis toxin (PTX, which abrogates Giα-coupled receptor signaling,FIG. 3 a), or high hypertonic sucrose solution (which inhibitsclathrin-coated pit dependent endocytosis, data not shown) completelyabolished ability of APCs to stimulate T cells. Similarly, chloroquine,the serine and cysteine protease inhibitor of lysosomal proteindegradation, or brefeldin A, a fungal metabolite that inhibits vesicletransport of newly synthesized MHC class molecules between theendoplasmic reticulum (ER) and Golgi²⁰, completely abrogated theresponse (FIG. 3 a), indicating the importance of lysosomal activity inthe chemoattractant-induced MHC class I presentation of OFA. Proteinswere shown to be processed directly within endosomal/lysosomalcompartments and loaded to MHC class I molecules, which resided inclassical MHC class II compartments, utilizing TAP-independent andNH₄Cl-sensitive cross-presentation pathways^(21,22). However, theCCR6-targeted OFA utilized classical cross-presentation pathway in thecytosol, since lactacystin, a specific inhibitor of proteasomal proteindegradation, completely abrogated the response (FIG. 3 a). Thepharmacological inhibitors used in this experiment did not causenon-specific suppressions, since they did not affect stimulation of Tcells induced by iDCs that were directly pulsed with OFA-iLRP₅₈₋₆₆peptide (that did not require internalization and processing, FIG. 3 a).These findings are supported by the confocal microscopy studiesdemonstrating that MIP3α-fusions, prior to internalization, werecolocalized with clathrin vesicles on the cell surface (0 min, FIG. 3b). However, within 10 min after internalization of MIP3α-fusions, theywere found in lyzosomes or colocalized with proteasomes in the cytosol(FIG. 3 b). The processed MIP3α-fusions were presumably degraded within1 hour after the internalization by lyzosomal enzymes and proteasomes(since the colocalized signal disappeared by 60 min incubation, FIG. 3b). Presumably, 60 min is sufficient to present processed peptides toMHC molecules, since iDCs incubated with MIP3α-OFA for as little as onehour were capable of stimulating immune T cells (though at much lowerlevels, data not shown). The peptides were presented onto H-2^(d)molecules, as the blocking antibody, but not control isotype matchedantibody, completely abolished ability of iDCs incubated with MIP3α-OFAor mDF2β-OFA to stimulate immune T cells (FIG. 3 c). Taken together,these data clearly demonstrate that potency of MIP3α-OFA or mDF2β-OFA isin their ability to use the CCR6-mediated uptake, processing andcross-presentation pathways. As a result, the vaccine elicited both CD4⁺T helper, as recently reported¹⁰, and cytolytic CD8⁺ T cell responsesleading to protection from A20 lymphoma, known for its resistance toimmunotherapy^(23;24). Thus, it is tempting to speculate that lack oftumor protection in mice immunized with OFA-iLRP₅₈₋₆₆ peptide/IFA (datanot shown) might be attributed to the absence of the T helper responses,although they generated CTLs capable of killing of A20 tumor cells invitro (FIG. 2 a).

Conclusion. The superiority of the CCR6-targeting OFA vaccines are intheir ability to elicit not only CD8⁺ CTLs (that recognized multiple OFAepitopes), but also in induction of Th1 helper CD4⁺ T cell responses.

Since this otherwise non-immunogenic OFA-iLRP is not expressed in normaladult tissues, the vaccine formulation can be also utilized as apreventive vaccine for induction of protective antitumor memoryresponses in healthy people at high risk for cancer.

Moreover, the vaccines of the invention have been shown to cause longlasting protective responses in mice.

Incorporation by Reference

The contents of all references, patents, pending patent applications andpublished patents, cited throughout this application are herebyexpressly incorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

REFERENCES

The following documents are referred to above and generally specified bysuperscript number corresponding to the reference number set forthbelow. Thus, for example, the first document of Cogin et al. AnticancerRes. 19:5535-5542 is referred to above with a superscript 1.

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1-29. (canceled)
 30. A polypeptide comprising a tumor-associatedembryonic antigen and a chemoattractant ligand.
 31. The polypeptide ofclaim 30, wherein the tumor-associated embryonic antigen is OFA-iLRP.32. The polypeptide of claim 30, wherein the chemoattractant ligand isspecific for CCR6.
 33. The nucleic acid molecule of claim 32, whereinthe chemoattractant ligand that is specific for CCR6 is MIP3α/CCL20 orβ-defensin mDF2β.
 34. The polypeptide of claim 33, wherein theMIP3α/CCL20 or β-defensin mDF2β is human or murine MIP3α/CCL20 orβ-defensin mDF2β.
 35. The polypeptide of claim 30, wherein thechemoattractant ligand is murine or human EP2C, human β-defensin 1(MBD1), or a C-terminal fragment of mycobacterial HSP
 70. 36. Thepolypeptide of claim 30, wherein the polypeptide comprises β-defensinDF2β and OFA-iLRP, or a functional fragment thereof. 37-38. (canceled)39. The polypeptide of claim 36, wherein the polypeptide has thesequence as set forth as SEQ ID NO:
 2. 40. The polypeptide of claim 30,wherein the polypeptide comprises MIP3α/CCL20and OFA-iLRP, or afunctional fragment thereof. 41-42. (canceled)
 43. The polypeptide ofclaim 40, wherein the polypeptide has the sequence as set forth as SEQID NO:
 4. 44. The polypeptide of claim 30, wherein the polypeptidecomprises EP2C and OFA-iLRP, or a functional fragment thereof. 45-46.(canceled)
 47. The polypeptide of claim 44, wherein the polypeptide hasthe sequence as set forth as SEQ ID NO:
 6. 48. The polypeptide of claim30, wherein the polypeptide comprises the C-terminal fragment ofmycobacterial HSP 70 and OFA-iLRP, or a functional fragment thereof. 49.The polypeptide of claim 48, wherein the polypeptide has the sequence asset forth as SEQ ID NO:
 8. 50-51. (canceled)
 52. The polypeptide ofclaim 30, further comprising a polypeptide linker between thetumor-associated embryonic antigen and the chemoattractant ligand.53-54. (canceled)
 55. A cancer vaccine comprising the polypeptide ofclaim
 30. 56. A method of treating a subject having cancer comprising;administering to the subject the polypeptide of claim 30; therebytreating the subject.
 57. (canceled)
 58. A method of immunizing asubject against cancer comprising: administering to the subject, thepolypeptide of claim 30, or the vaccine of claim 54; thereby immunizingthe subject.
 59. A kit comprising the vaccine of claim 54 andinstructions for use. 60-61. (canceled)